COVID-19 and Parkinson’s disease: a single-center study and Mendelian randomization study

To investigate the association between COVID-19 and Parkinson’s disease (PD) via a single-center study and a Mendelian randomization (MR) study. A questionnaire-based survey was conducted among PD patients at a single center from December 7, 2022, to March 10, 2023. Logistic regression analysis was performed to identify the infection-related risk factors. Subsequently, bidirectional two-sample Mendelian randomization was employed to explore the association between COVID-19 and PD. In the cross-sectional analysis, it was found that the prevalence of COVID-19 infection in PD patients was 65.7%. Forty-eight (35.3%) PD patients experienced exacerbation of motor symptoms following COVID-19 infection. Long PD disease duration (≥ 10 years) (OR: 3.327, P = 0.045) and long time since last vaccination (> 12 m) (OR: 4.916, P = 0.035) were identified as significant risk factors related to infection. The MR analysis results supported that PD increases the COVID-19 susceptibility (β = 0.081, OR = 1.084, P = 0.006). However, the MR analysis showed that PD did not increases the COVID-19 severity and hospitalization, and no significant association of COVID-19 on PD was observed. The findings from this cross-sectional study suggest that individuals with PD may experience worsened motor symptoms following COVID-19 infection. Long disease duration (≥10 years) and long time since last vaccination (> 12 m) are identified as important risk factors for infection in these patients. Furthermore, our MR study provides evidence supporting an association between PD and COVID-19 susceptibility.

www.nature.com/scientificreports/COVID-19 was described by Calculli A et al. 8 .Furthermore, studies have indicated that the mortality rate of COVID-19 in PD patients surpasses that of the general elderly population 9,10 .However, one study showed that available data did not yet justify a clear association between the COVID-19 pandemic and a parkinsonism wave 11 .Therefore, comprehending the intricate relationship between COVID-19 and PD is crucial for devising effective interventions to improve patient outcomes.
People with PD are recommended to receive the COVID-19 vaccine, unless they have specific contraindications 12 .A study has indicated a low vaccination rate among patients with PD 12 .However, limited information is available regarding the COVID-19 vaccination status and its protective effect on these individuals.
The present study aimed to determine the effect on patients with PD after the release of COVID-19 restrictions and analyze the infection-related risk factors of these patients, as well as evaluate the protective effect of COVID-19 vaccination on PD through a single-center study.Mendelian randomization (MR) is an effective genetic method to study the causal relationship of certain exposures to disease.Therefore, we additionally conducted a bidirectional two-sample MR analysis to explore the potential causal correlation between COVID-19 and PD.

Cross-sectional study design
This study had a cross-sectional design.The inclusion criteria were as follows: (1) patients diagnosed with PD according to the MDS clinical diagnosis criteria and (2) patients who maintained their medication regimen without any changes within 1 week prior to December 7, 2022.The exclusion criteria included PD patients who were unable to complete the questionnaire or lacked detailed information.Questionnaires were collected from patients who met the inclusion and exclusion criteria, who visited the outpatient and inpatient departments at the Affiliated Hospital of Guizhou Medical University between December 7, 2022, and March 10, 2023.
First, the respondents were informed that participation in the survey was greatly appreciated, that only anonymous data were collected.Second, clinical data was obtained via an interview and structured questionnaire after obtaining informed consent.The questionnaire included demographic information (age and sex), COVID-19 vaccination history (duration and type of last vaccination),PD-related clinical data (motor and non-motor symptoms), clinical features of COVID-19, as well as alterations in PD symptoms after COVID-19 infection.Third, questionnaires were completed by patients with PD themselves or with assistance from their family members if they faced difficulties in reading or writing.Trained neurologists specialized in PD administered the questionnaire.In this study, COVID-19 infection was defined as exhibiting symptoms of COVID-19 along with a positive result for COVID-19 nucleic acid or antigen between December 7, 2022, and January 7, 2023.However, symptomatic patients with negative laboratory results and asymptomatic individuals who had not been tested for COVID-19 nucleic acid or antigen were excluded from the definition of COVID-19 infection.

Study design
In this study, we primarily used a bidirectional two-sample MR method.The MR study relies on three fundamental assumptions: firstly, there should exist a robust association between genetic variation and the exposure factor (association assumption).Secondly, the exposure factor should not possess any direct relationship with the outcome (exclusion assumption).Lastly, genetic variation should be independent of any potential confounding factors (independence assumption).The process is illustrated in Fig. 1, which presents the relevant flowchart.

Data sources
We used available data on COVID-19 phenotypes from the COVID-19 Host Genetics Project (RELEASE 5) based on a European population 13 .The comprehensive dataset comprises information regarding susceptibility,  The PD dataset analyzed in this study was derived from a large GWAS meta-analysis published by International Parkinson's Disease Genomics Consortium 14 .The study was also based on a European population of 482,730 participants, including 33,674 cases of PD and 449,056 healthy controls (Table 1).PD cases in this study were defined based on the UK brain bank criteria from clinic visit, or the use of PD medication or medical records of PD diagnosis by clinician, or via study participant reports of the diagnosis of PD.

Selection of instrumental variables
The instrumental variables (single nucleotide polymorphisms, SNPs) were carefully selected based on their strong correlation with exposure (P < 5 × 10 −8 ) and pruned by linkage disequilibrium (r 2 < 0.001 and within 10,000 kb from the index variant).Outlier SNPs were identified and removed using MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) 15 .We examined whether the obtained instrumental SNPs were associated with the outcomes and the potential confounders by PhenoScanner (http:// www.pheno scann er.medsc hl.cam.ac.uk/).The F-statistics of these SNPs were employed to evaluate the strength of each instrumental variable.The R 2 value for each SNP was calculated using the formula: R 2 = 2 × EAF × (1 − EAF) × β 216 .

MR analysis and sensitivity analysis
To determine whether there is a causal association between COVID-19 and PD, we mostly employ the inverse variance weighted (IVW) method for MR analysis.Additionally, complementary methods such as MR-Egger, weighted median, simple mode, and weighted mode were utilized in conjunction with IVW.The Cochran's Q test of the IVW approach and leave-one-out analysis were used to investigate the degree of heterogeneity.The MR-Egger intercept test and MR-PRESSO global test were employed to evaluate the horizontal pleiotropy.

Statistical analysis
Statistical analyses were performed using SPSS statistical software for Windows version 24.0 (SPSS, Chicago, IL, USA) for cross-sectional study.The independent sample t-test was employed to analyze continuous variables with normal distributions, including general data and clinical basic information.Chi-square test and Fisher exact test were used to compare categorical variables.Logistic regression analysis was performed to analyze the infection-related risk factors.A significance level of P < 0.05 indicated statistical significance.
For MR analysis, all analyses were carried out in R (version 4.3.0)utilizing the TwoSample MR and MR-PRESSO packages [15][16][17] .P < 0.05 was considered as nominally significant and P < 0.05/6 (0.008) was considered as significant after correcting by Bonferroni measures.

Ethics approval
The protocol for the research project has been approved by a suitably constituted Ethics Committee of the institution within which the work was undertaken and that it conforms to the provisions of the Declaration of Helsinki (as revised in Fortaleza, Brazil, October 2013).

Approval of the research protocol
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Affiliated Hospital of Guizhou Medical University.

Informed consent
All participants provided informed consent before enrollment.

Characteristics of patients with PD
In the period of study, a total of 258 patients with PD were admitted to our hospital.Among them, 209 patients fulfilled the inclusion and exclusion criteria and were invited to participate in this study.Two patients were excluded due to insufficient information provided.Finally, a total of 207 PD patients participated in this study.
The baseline characteristics and clinical features of both PD and COVID-19 are presented in Table 2.A total of 106 male and 101 female participants were enrolled in this study.The mean age of the participants was 67.76 years (SD ± 9.71, range 39-88), with a mean PD duration of 5.24 years.Among the PD patients, a total of 136 individuals were infected with COVID-19, resulting in an infection rate of 65.7%.The mean ages of the patients infected or uninfected with COVID-19 were 66.83 (SD ± 11.26, range 39-84) and 68.58 (SD ± 9.75, range 46-88) years, respectively (P = 0.269).No significant differences were observed between the COVID-19-infected and uninfected groups regarding sex (P = 0.917), courses of the PD (P = 0.458), vaccination status (P = 0.663), rest tremor (P = 0.974), bradykinesia (P = 0.469), rigidity (P = 0.131), or posture instability (P = 0.534).However, the result revealed a significant difference between the COVID-19-infected and uninfected groups in terms of time since last COVID-19 vaccination (P < 0.01).
Forty-eight patients experienced a worsening of motor symptoms, resulting in an exacerbation rate of 35.3%.The deteriorated motor symptoms included rest tremor (N = 14), bradykinesia (N = 16), rigidity (N = 12), posture instability (N = 10), wearing-off (N = 8), weakness (N = 8), and dyskinesia (N = 1) (Fig. 3).During the questionnaire period, it remained uncertain whether the worsening of PD symptoms was transient or permanent, necessitating further monitoring of symptom progression in these patients at subsequent stages.Sixteen (11.8%) patients with PD were admitted to hospitals due to COVID-19 infection, and one fatality occurred as a result of respiratory failure.More than half of the hospitalized patients had an average hospitalization duration of one week.3).Notably, individuals who received their COVID-19 vaccine within three months exhibited a lower infection rate compared to those vaccinated after three months (50% vs. 67.3%).These results suggest that the COVID-19 vaccine provides short-term protection for patients with PD.
The existence of gene pleiotropy was tested using MR-Egger regression analysis, and all intercept terms were found to be close to zero (P > 0.05).The MR-PRESSO method was also employed and yielded consistent results with the MR-Egger regression.Although the P-values of the MR-PRESSO analysis was less than 0.05 in COVID-19 severity, horizontal pleiotropy did not present in the result of MR-PRESSO destruction test (P > 0.05).Furthermore, Cochran's statistical test revealed no significant heterogeneity effect (Q-value > 0.05) (Table 4).

Discussion
The rapid spread of the COVID-19 pandemic poses particular challenges for the management of patients with PD 18 .One study showed that mortality and hospitalization rates among elderly individuals with PD did not significantly differ from those of the general population 18 .However, another study revealed a higher COVID-19 mortality rate among PD patients compared to other hospitalized individuals 19 .Hence, the association between COVID-19 and PD remains unclear.Furthermore, on December 7, 2022, China introduced "10 new measures" to adjust strategies for preventing and controlling COVID-19.Therefore, further investigation is required to identify the relationship between COVID-19 and PD and develop future intervention measures.
The findings of one study revealed that within 3 weeks after the relaxation of COVID-19 restrictions in Macao, China, the omicron variant epidemic exhibited a staggering infection rate of 75% 3 .Additionally, another study indicated that the omicron epidemic reached an infection rate exceeding 70%, approximately one week earlier than observed in Macao 3 .These studies provide suggestive evidence regarding post-restriction COVID-19 infections.Furthermore, our present study demonstrated the rapid transmission of the omicron variant among patients with PD, with an infection rate of 65.7% within a month following the release of COVID-19 restrictions in China.
One study revealed that over 60 percent of individuals infected with COVID-19 exhibited symptoms such as fever, dry or sore throat, nasal congestion and runny nose, fatigue, headaches, or muscle aches 3 .Similarly, the predominant COVID-19 symptoms observed in patients with PD in this study included coughing, fever, fatigue, dry or sore throat, nasal congestion or runny nose, myalgia, dizziness and headache along with gastrointestinal symptoms.More than half of PD patients experienced COVID-19 symptoms within one week.
The effect of COVID-19 on patients with PD is multifaceted.Several studies have indicated that COVID-19 infection may exacerbate both motor and non-motor symptoms of PD, or give rise to previously unobserved symptoms 20,21 .A study presented two patients with PD who underwent subthalamic deep brain stimulation and experienced rapid deterioration in their PD symptoms subsequent to contracting COVID-19 22 .In this current study, over one-third of the patients with PD exhibited aggravated motor symptoms, with bradykinesia being the most prominent.However, it remained unclear during the questionnaire period whether these worsened PD symptoms were transient or permanent, necessitating further monitoring of symptom evolution in these patients at a later stage.Studies have suggested that COVID-19 infection may aggravate PD symptoms through potential mechanisms such as hematologic pathways or axonal transport via olfactory neuroepithelium, leading to inflammation, immunologically mediated mitochondrial injury, and neuronal oxidative stress 8,23 .One study reported an increased risk ratio (RR) of developing PD 6 and 12 months after a positive COVID-19 test compared to individuals who tested negative for COVID-19 24 .However, it is important to note that any intercurrent illness (e.g.flu, urinary infection, pneumonia) can temporarily exacerbate PD symptoms.Further research is needed to compare the exacerbation of PD symptoms between COVID-19 and other concurrent illnesses.Therefore, we performed a two-sample MR analysis to determine the association between COVID-19 and PD.However, we found no statistically significant effect of COVID-19 susceptibility, hospitalization and severity on the increased risk of PD at the genetic level.Nevertheless, it is important to acknowledge that this analysis may be constrained by data limitations, necessitating further cohort studies to explore this relationship.
Vaccination is a highly effective measure for preventing severe illness caused by COVID-19 and reducing the transmission of infection.A study revealed that the COVID-19 vaccination rate among patents with PD was 54.0% 12 .However, in our present study, the COVID-19 vaccination rate was found to be higher at 80.2% compared to the previous study.Furthermore, our results indicated that the long time since last vaccination (> 12 m) was one of the infection-related risk factors of patients with PD.Additionally, it was observed that the COVID-19 vaccine provided short-term protection for individuals with PD.Therefore, we recommend COVID-19 vaccination for PD patients unless there are specific contraindications.
There was no significant difference in the risk of COVID-19 infection between PD patients and healthy controls during the COVID-19 pandemic according to one study 18 , indicating that PD is not a contributing factor to the susceptibility of COVID-19.However, another study showed that PD patients possess a higher rate of COVID-19 mortality 19 .The findings of the present study showed that long disease duration (≥ 10 years) was another infection-related risk factor.Therefore, we further determine the association between PD and COVID-19, and our MR study provided evidence supporting a significant association between PD and an elevated susceptibility to COVID-19.However, we did not find any statistically significant effect of PD on the increased risk of hospitalization or severity related to COVID-19 at the genetic level.
Due to the change in China's current COVID-19 management policy, understanding the effect on patients with PD after the release of COVID-19 restrictions and taking timely preventive measures in the future, such as administrating booster vaccine and stockpiling antiviral drugs, could reduce the morbidity and mortality of these patients in the future epidemic wave.
There were several limitations to our study.Firstly, COVID-19 infection in our study was defined as the presence of respiratory symptoms along with positive nucleic acid tests or antigen detection.However, it is important to acknowledge that asymptomatic individuals who did not undergo nucleic acid or antigen testing were not included in our study cohort, potentially leading to an underestimation of the true infection rate.Secondly, the analysis did not differentiate PD disease severity separately, which may result in overlooking distinctions between PD disease severity and COVID-19.Thirdly, it remains unclear whether the worsening of PD symptoms was transient or permanent.Lastly, it should be noted that this study did not investigate the association between previous COVID-19 infections and PD within different timeframes due to data limitations.Therefore, additional cohort studies are required to investigate this relationship.

Conclusions
More than one-third of patients with PD exhibits exacerbated motor symptoms, with bradykinesia being the most prominent.The findings from this cross-sectional study suggest that COVID-19 infection contributes to the deterioration of motor symptoms in PD patients.Notably, long disease duration (≥ 10 years) and long time since last vaccination (> 12 m) are identified as risk factors associated with infection.Furthermore, the MR study supports the association between PD and a higher susceptibility to COVID-19.In addition, PD patients are encouraged to be receive booster vaccine because of the short-term protective effect of COVID-19 vaccine on them.

Figure 1 .
Figure 1.The overview of MR analysis in the study.A1: SNPs are robustly associated with exposures.A2: SNPs are not associated with confounders.A3: SNPs affect the outcome only through the exposures and not through other pathways.
www.nature.com/scientificreports/Infection-related risk factors and protective effect of COVID-19 vaccine A total of 166 patients with PD were vaccinated against COVID-19, with a vaccination rate of 80.2%.Logistic regression analysis was conducted to investigate the infection-related risk factors in these patients, considering age, sex, course of PD, time since last vaccination, and type of the last dose of vaccination as independent variables.The findings revealed that long disease duration (≥ 10 years) (OR = 3.327, 95% CI 1.029-10.755)and long time since last vaccination (> 12 m) (OR = 4.916, 95% CI 1.116-21.650)were identified as significant risk factors (Table

Figure 4 .
Figure 4. MR estimates for the causality of the association between COVID-19 and PD.Panels (A-C) respectively indicate the causal estimates of COVID-19 hospitalization, severity, and susceptibility on PD.Panels (D-F) respectively indicate the causal estimates of PD on COVID-19 hospitalization, severity, and susceptibility.

Figure 5 .
Figure 5. Scatter plot of the MR estimate for the effect of PD on the risk of COVID-19 susceptibility.
hospitalization, and severity of COVID-19.The susceptibility phenotype involved a comparison between COVID-19 patients and controls without COVID-19 (Ncase = 38,984, Ncontrol = 1,644,784).The hospitalization phenotype compared hospitalized COVID-19 patients with a control group that was neither hospitalized for nor infected with COVID-19 (Ncase = 9986, Ncontrol = 1,877,672).The severity phenotype compared hospitalized COVID-19 patients who died or required respiratory support with a control group without severe COVID-19 or free of COVID-19 (Ncase = 5101, Ncontrol = 1,383,241) (Table 1).In this study, COVID-19 susceptibility was diagnosed in accordance with the WHO interim guidance, or Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 6), National Health Commission of the People's Republic of China.

Table 2 .
The basic characteristics of participants (n = 207).*Independent sample t-test was used for the analyses of continuous variables and had normal distributions.†Chi-square test was used to compare categorical variables.Significant values are in bold.

Table 4 .
Mendelian randomization estimates for associations between COVID-19 and PD.CI confidence interval, IVW inverse variance-weighted, P-heterogeneity P-value for heterogeneity using Cochran's Q test, P-intercept P-value for MR-Egger intercept, P-PRESSO P-value for MR-PRESSO global test.*P value is significant.